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Journal of Enzyme Inhibition and Medicinal Chemistry
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7-Acylamino-3H-1,2-benzoxathiepine 2,2-dioxides
as new isoform-selective carbonic anhydrase IX
and XII inhibitors
Aleksandrs Pustenko , Alessio Nocentini , Anastasija Balašova , Mikhail
Krasavin , Raivis Žalubovskis & Claudiu T. Supuran
To cite this article:
Aleksandrs Pustenko , Alessio Nocentini , Anastasija Balašova , Mikhail
Krasavin , Raivis Žalubovskis & Claudiu T. Supuran (2020) 7-Acylamino-3H-1,2-benzoxathiepine
2,2-dioxides as new isoform-selective carbonic anhydrase IX and XII inhibitors, Journal of Enzyme
Inhibition and Medicinal Chemistry, 35:1, 650-656, DOI: 10.1080/14756366.2020.1722658
To link to this article: https://doi.org/10.1080/14756366.2020.1722658
© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Published online: 21 Feb 2020.
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RESEARCH PAPER
7-Acylamino-3H-1,2-benzoxathiepine 2,2-dioxides as new isoform-selective
carbonic anhydrase IX and XII inhibitors
Aleksandrs Pustenko
a,b, Alessio Nocentini
c, Anastasija Bala
sova
a, Mikhail Krasavin
d, Raivis
Zalubovskis
a,band
Claudiu T. Supuran
ca
Latvian Institute of Organic Synthesis, Riga, Latvia;bInstitute of Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, Latvia;cDipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Florence, Italy;dDepartment of Chemistry, Saint Petersburg State University, Saint Petersburg, Russian Federation
ABSTRACT
A series of 3H-1,2-benzoxathiepine 2,2-dioxides incorporating 7-acylamino moieties were obtained by an original procedure starting from 5-nitrosalicylaldehyde, which was treated with propenylsulfonyl chloride followed by Wittig reaction of the bis-olefin intermediate. The new derivatives, belonging to the homosul-focoumarin chemotype, were assayed as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Four pharmacologically relevant human (h) isoforms were investigated, the cytosolic hCA I and II and the transmembrane, tumour-associated hCA IX and XII. No relevant inhibition of hCA I and II was observed, whereas some of the new derivatives were effective, low nanomolar hCA IX/XII inhibitors, mak-ing them of interest for investigations in situations in which the activity of these isoforms is overex-pressed, such as hypoxic tumours, arthritis or cerebral ischaemia.
ARTICLE HISTORY Received 26 December 2019 Revised 21 January 2020 Accepted 23 January 2020 KEYWORDS Carbonic anhydrase; transmembrane isoforms; sulfocoumarin; homosulfo-coumarin; isoform-selective inhibitor
1. Introduction
Sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) and homosulfo-coumarins (3H-1,2-benzoxathiepine 2,2-dioxides)1–5 are among the most investigated new classes of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, which have been designed considering the structurally similar coumarins6–8as lead molecules. Indeed, Cas are widely spread enzymes in organisms of all types, from simple to complex ones9–15, and are involved in crucial physiological proc-esses, among which carbon fixation in diatoms and other marine organisms in which several genetic families of such metalloen-zymes were reported9. In protozoans, Cas are involved in biosyn-thetic reactions9 whereas in bacteria, where at least three genetic families were described (a-, b-, and c-Cas) these enzymes play cru-cial roles related both to metabolism but also virulence and sur-vival in various niches10. In vertebrates, including humans, a high number of different CA isoforms belonging to the a-CA class were described11,12, which by hydrating CO2to a weak base (bicarbon-ate) and a strong acid (hydronium ions), are involved in a multi-tude of processes, starting with pH regulation and ending with metabolism13,14. As thus, Cas are drug targets for decades, with their inhibitors having pharmacological applications in a multitude of fields11–16. The primary sulphonamides were discovered as CA inhibitors (CAIs) in the ‘40 s, and most of the drugs that were launched in the next decades as diuretics, antiepileptics, or anti-glaucoma agents belonged to this class of compounds or to their isosteres such as the sulfamates and sulfamides11. An important drawback of such first generation CA inhibitors (CAIs) was their lack of isoform selectivity, considering the fact that in humans at
least 12 catalytically active and three acatalytic isoforms are pre-sent11,12. However, the new generation CAIs to which coumarins
and sulfocoumarins belong, show significant isoform-selective inhibition profiles, as demonstrated in a considerable number of studies1–8. This is principally due to the fact that these com-pounds possess a distinct inhibition mechanism compared to the sulphonamides, which coordinate to the zinc ion from the CA active site as anions11,12. In fact, coumarins and sulfocoumarins act as prodrug inhibitors, undergoing an active site mediated hydrolysis, which leads to the formation of 2-hydroxy-cinnamic acids in the case of the coumarins, and ethane-sulphonates in the case of the sulfocoumarins, which subsequently bind in different active site regions, different of those where the classical sulphona-mide CAIs bind1–8. As shown by X-ray crystallography, the hydro-lysed coumarins occlude the entrance of the CA active site cavity6, whereas the sulfocoumarins bind deeper within the active
site, but still do not coordinate to the metal ion. Instead, the formed sulphonates anchor to the zinc-coordinated water mol-ecule, as shown again by means of X-ray crystallographic techni-ques2. As these regions of the CA active site are the most variable ones, a straightforward explanation of the isoform selectivity of these new generation CAIs was furnished by using a combination of crystallographic and kinetic studies, which also allowed the development of compounds showing a higher degree of selectiv-ity15,16. This allowed for the development of inhibitors useful for new pharmacological applications such as antitumor/antimeta-static compounds13, CAIs useful for the management of arthritis17, neuropathic pain18, and cerebral ischaemia19.
CONTACT Raivis Zalubovskis raivis@osi.lv Latvian Institute of Organic Synthesis, 21 Aizkraukles Str, Riga, LV-1006, Latvia; Claudiu T. Supuran
claudiu.supuran@unifi.it Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Sesto Fiorentino,
Florence, Italy
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2020, VOL. 35, NO. 1, 650–656
Considering our interest in designing non-sulphonamide CAIs with various potential applications, we report here a new series of homosulfocoumarins and their inhibitory profiles against the major human (h) CA isoforms, hCA I, II, IX, and XII, involved in many pathologies, including cancer.
2. Experimental part
2.1. ChemistryReagents, starting materials/intermediates 1–7 and solvents were obtained from commercial sources (Sigma-Aldrich, St. Louis, MO) and used as received. Anhydrous CH2Cl2 and toluene were obtained by passing commercially available solvents through acti-vated alumina columns. Thin-layer chromatography was per-formed on silica gel, spots were visualised with UV light (254 and 365 nm). Melting points were determined on an OptiMelt auto-mated melting point system. IR spectra were recorded on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Bruker Avance Neo (400 MHz) spectrometer with chemical shifts values (d) in ppm relative to TMS using the residual DMSO-d6 signal (1H 2.50; 13C 39.52) or CDCl3 signal (1H 7.26; 13C 77.16) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyser using the ESI technique.
General procedure for synthesis of acyl compound 8–17
To a solution of amino derivative 7 (1.0 eq.) in dry CH2Cl2 (20 ml per mmol of compound 7) at 0C appropriate acyl chloride (1.1 eq.) and Net3 (1.1 eq.) were added. The resulting mixture was stirred at room temperature under an argon atmosphere for 2 h. Water was added (20 ml per mmol of compound 7). Layers were separated, water layer was washed with EtOAc (2 40 ml). Combined organic layers were washed with brine, dried over anh. Na2SO4, filtered, evaporated. The crude solids were recrystallised form EtOAc/petrol ether mixture to afford product.
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)acetamide (8).
O SO2
H N O
Compound 8a was prepared according to the general procedure from amino deriva-tive 7 (150 mg; 0.71 mmol), acetyl chloride (56mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (127 mg; 70%). Mp 164–165C.
IR (film, cm1) max¼ 3276 (N–H), 1670 (C ¼ O), 1370 (S ¼ O), 1361 (S¼ O), 1166 (S ¼ O), 1162 (S ¼ O);
1H NMR (400 MHz, DMSO-d 6) d¼ 2.06 (s, 3H), 4.37–4.41 (m, 2H), 5.96–5.6.04 (m, 1H), 6.89 (d, 1H, J ¼ 11.3 Hz), 7.28 (d, 1H, J ¼ 8.9 Hz), 7.58 (dd, 1H, J ¼ 8.9, 2.5 Hz), 7.69 (d, 1H, J ¼ 2.5 Hz), 10.16 (s, 1H) ppm. 13 C NMR (100 MHz, DMSO-d6) d¼ 24.0, 51.0, 120.6, 120.8, 122.7, 128.4, 131.5, 138.0, 142.2, 168.6 ppm.
HRMS (ESI) [Mþ H]þ: m/z calcd for (C11H12NO4S) 254.0487. Found 254.0498. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)benzamide (9). O O SO2 H N
Compound 9 was prepared according to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), ben-zoyl chloride (90mL; 0.78 mmol) and
Et3N (110mL; 0.78 mmol) as white solid (162 mg; 72%). Mp 174–175C.
IR (film, cm1) max¼ 3289 (N–H), 1652 (C ¼ O), 1370 (S ¼ O), 1363 (S¼ O), 1163 (S ¼ O); 1 H NMR (400 MHz, DMSO-d6) d¼ 4.43 (dd, 2H, J ¼ 6.0, 0.9 Hz), 5.99–6.06 (m, 1H), 6.93 (d, 1H, J ¼ 11.2 Hz), 7.35 (d, 1H, J ¼ 8.8 Hz), 7.52–7.58 (m, 2H), 7.59–7.64 (m, 1H), 7.82 (dd, 1H, J ¼ 8.8, 2.5 Hz), 7.91 (d, 1H,J ¼ 2.5 Hz), 7.94–7.99 (m, 2H), 10.46 (s, 1H) ppm. 13 C NMR (100 MHz, DMSO-d6) d¼ 51.1, 120.9, 122.0, 122.1, 122.6, 127.7, 128.3, 128.5, 131.4, 131.8, 134.6, 137.9, 142.7, 165.7 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C16H14NO4S) 316.0644. Found 316.0654. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-4-methy benzamide (10). O SO2 H N O
Compound 10 was prepared according to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 4-methylbenzoyl chlor-ide (103mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white crys-tals (170 mg; 73%). Mp 197–198C.
IR (film, cm1) max ¼ 3324 (N–H), 1646 (C ¼ O), 1378 (S ¼ O), 1363 (S¼ O), 1177 (S ¼ O), 1169 (S ¼ O);
1 H NMR (400 MHz, DMSO-d6) d¼ 2.39 (s, 3H), 4.41–4.45 (m, 2H), 5.99–6.06 (m, 1H), 6.92 (d, 1H, J ¼ 11.2 Hz), 7.32–7.37 (m, 3H), 7.82 (dd, 1H,J ¼ 8.9, 2.6 Hz), 7.86–7.92 (m, 3H), 10.37 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 21.0, 51.1, 120.8, 121.9, 122.1, 122.6, 127.7, 128.3, 129.0, 131.4, 131.6, 138.0, 141.9, 142.6, 165.5 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C17H16NO4S) 330.0800. Found 330.0815. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)4-bromobenzamide (11). O O Br SO2 H N
Compound 11 was prepared according to the general proced-ure from amino derivative 7 (150 mg; 0.71 mmol), 4-bromoben-zoyl chloride (171 mg; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (166 mg; 59%). Mp 185–186C.
IR (film, cm1) max¼ 3260 (N–H), 1653 (C ¼ O), 1375 (S ¼ O), 1363 (S¼ O), 1167 (S ¼ O); 1H NMR (400 MHz, DMSO-d 6) d¼ 4.42–4.46 (m, 2H), 5.99–6.06 (m, 1H), 6.92 (d, 1H,J ¼ 11.3 Hz), 7.35 (d, 1H, J ¼ 8.8 Hz), 7.74–7.83 (m, 3H), 7.88–7.94 (m, 3H), 10.52 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 51.2, 120.9, 122.0, 122.2, 122.7, 125.6, 128.3, 129.8, 131.4, 131.5, 133.6, 137.7, 142.8, 164.7 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C16H13BrNO4S) 393.9749. Found 393.9736. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-iodobenzamide (12). O SO2 H N O I
Compound 12 was prepared according to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 2-iodobenzoyl chloride (208 mg; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (276 mg; 88%). Mp 188–189C.
IR (film, cm1) max¼ 3240 (N–H), 1641 (C ¼ O), 1374 (S ¼ O), 1362 (S¼ O), 1156 (S ¼ O);
1 H NMR (400 MHz, DMSO-d6) d¼ 4.41–4.45 (m, 2H), 6.00–6.08 (m, 1H), 6.94 (d, 1H, J ¼ 11.2 Hz), 7.22–7.28 (m, 1H), 7.36 (d, 1H, J ¼ 8.8 Hz), 7.47–7.55 (m, 2H), 7.72 (dd, 1H, J ¼ 8.8, 2.5 Hz), 7.87 (d, 1H,J ¼ 2.5 Hz), 7.9–7.97 (m, 1H), 10.67 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 51.0, 93.6, 121.0, 121.2, 121.3, 122.9, 128.1, 128.2, 128.5, 131.2, 131.5, 137.7, 139.1, 142.7, 142.8, 167.7 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C16H13INO4S) 441.9610 Found 441.9609. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-bromobenzamide (13). O SO2 H N O Br
Compound 13 was prepared accord-ing to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 2-bromobenzoyl chloride (102mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (230 mg; 82%). Mp 177–178C.
IR (film, cm1) max¼ 3288 (N–H), 1653 (C ¼ O), 1371 (S ¼ O), 1176 (S¼ O), 1156 (S ¼ O); 1 H NMR (400 MHz, DMSO-d6) d¼ 4.43 (dd, 2H, J ¼ 6.0, 0.9 Hz), 6.00–6.07 (m, 1H), 6.94 (d, 1H, J ¼ 11.2 Hz), 7.36 (d, 1H, J ¼ 8.9 Hz), 7.41–7.47 (m, 1H), 7.51 (dt, 1H, J ¼ 7.4, 1.1 Hz), 7.55–7.59 (m, 1H), 7.69–7.76 (m, 2H), 7.87 (d, 1H, J ¼ 2.6 Hz), 10.73 (s, 1H) ppm 13C NMR (100 MHz, DMSO-d 6) d¼ 51.0, 118.9, 121.1, 121.2, 122.9, 127.8, 128.6, 128.9, 131.4, 131.5, 132.8, 137.6, 138.8, 142.8, 166.0 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C16H13BrNO4S) 393.9749 Found 393.9766. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-fluorobenzamide (14). O O SO 2 H N F
Compound 14 was prepared accord-ing to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 2-fluorobenzoyl chloride (93mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (188 mg; 79%). Mp 173–174C.
IR (film, cm1) max¼ 1671 (C ¼ O), 1371 (S ¼ O), 1365 (S ¼ O), 1164 (S¼ O) 1156 (S ¼ O); 1 H NMR (400 MHz, DMSO-d6) d¼ 4.41–4.45 (m, 2H), 6.00–6.07 (m, 1H), 6.93 (d, 1H,J ¼ 11.2 Hz), 7.32–7.40 (m, 3H), 7.56–7.63 (m, 1H), 7.65–7.71 (m, 1H), 7.74 (dd, 1H, J ¼ 8.8, 2.5 Hz), 7.86 (d, 1H, J ¼ 2.5 Hz), 10.65 (s, 1H) ppm 1. 13C NMR (100 MHz, DMSO-d6) d¼ 51.1, 116.2 (d, J ¼ 21.7 Hz), 121.0, 121.4, 121.5, 122.8, 124.6 (d, J ¼ 5.5 Hz), 124.7 (d, J ¼ 6.3 Hz), 128.5, 129.9 (d, J ¼ 2.6 Hz), 131.4, 132.8 (d, J ¼ 8.5 Hz), 137.5, 142.8, 159.9 (d, J ¼ 249 Hz), 163.0 ppm 2. HRMS (ESI) [Mþ H]þ: m/z calcd for (C16H13FNO4S) 334.0549
Found 334.0554. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-(trifluoromethyl)ben-zamide (15). O SO2 H N O CF3
Compound 15 was prepared accord-ing to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 2-(trifluoromethyl)ben-zoyl chloride (115mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (236 mg; 87%). Mp 192–193C.
IR (film, cm1) max¼ 3195 (N–H), 1666 (C ¼ O), 1377 (S ¼ O), 1316 (S¼ O), 1164 (S ¼ O); 1 H NMR (400 MHz, DMSO-d6) d¼ 4.42–4.45 (m, 2H), 6.00–6.08 (m, 1H), 6.94 (d, 1H,J ¼ 11.2 Hz), 7.36 (d, 1H, J ¼ 8.8 Hz), 7.67–7.76 (m, 3H), 7.78–7.89 (m, 3H), 10.81 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 51.0, 121.1, 121.3, 122.9, 123.8 (q, J ¼ 274 Hz), 125.8 (q, J ¼ 31.2 Hz), 126.4 (q, J ¼ 4.6 Hz), 128.5, 128.6, 130.3, 131.4, 132.7, 135.8 (q, J ¼ 2.3 Hz), 137.6, 142.8, 165.8 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C17H13NO4 F3S) 384.0517 Found 384.0519. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)thiophene-2-carboxa-mide (16). O SO2 H N O S
Compound 16 was prepared accord-ing to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 2-thiophenecarbonyl chloride (84mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (185 mg; 81%). Mp 162–163C.
IR (film, cm1) max¼ 3357 (N–H), 1648 (C ¼ O), 1372 (S ¼ O), 1356 (S¼ O), 1178 (S ¼ O), 1165 (S ¼ O);
1 H NMR (400 MHz, DMSO-d6) d¼ 4.44 (dd, 2H, J ¼ 6.0, 1.1 Hz), 5.99–6.06 (m, 1H), 6.92 (d, 1H, J ¼ 11.2 Hz), 7.23–7.26 (m, 1H), 7.35 (d, 1H, J ¼ 8.8 Hz), 7.78 (dd, 1H, J ¼ 8.8, 2.6 Hz), 7.84 (d, 1H, J ¼ 2.6 Hz), 7.88 (dd, 1H, J ¼ 5.0, 1.1 Hz), 8.04 (dd, 1H, J ¼ 3.8, 1.1 Hz), 10.43 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 51.2, 120.9, 121.9, 122.1, 122.7, 128.2, 128.4, 129.5, 131.3, 132.3, 137.5, 139.5, 142.7, 160.0 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C14H12NO4S2) 322.0208 Found 322.0221. N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)furan-2-carboxamide (17). O SO2 H N O O
Compound 17 was prepared according to the general procedure from amino derivative 7 (150 mg; 0.71 mmol), 2-furoyl chloride (84mL; 0.78 mmol) and Et3N (110mL; 0.78 mmol) as white solid (185 mg; 81%). Mp 162–163C.
IR (film, cm1) max¼ 3299 (N–H), 1663 (C ¼ O), 1367 (S ¼ O), 1363 (S¼ O), 1165 (S ¼ O), 1158 (S ¼ O);
1 H NMR (400 MHz, DMSO-d6) d¼ 4.41–4.45 (m, 2H), 5.98–6.06 (m, 1H), 6.70–6.74 (m, 1H), 6.90 (d, 1H, J ¼ 11.2 Hz), 7.32–7.38 (m, 2H), 7.80 (dd, 1H,J ¼ 8.8, 2.6 Hz), 7.87 (d, 1H, J ¼ 2.6 Hz), 7.95–7.97 (m, 1H), 10.41 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 51.2, 112.3, 115.2, 120.9, 122.0, 122.2, 122.6, 128.3, 131.4, 137.3, 142.7, 146.0, 147.2, 156.3 ppm
HRMS (ESI) [Mþ H]þ: m/z calcd for (C14H12NO5S) 306.0436 Found 306.0463.
2.2. CA inhibitory assay
An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2hydration activity
20
. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO2 hydra-tion reachydra-tion for a period of 10 100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5 10% of the reaction have been used for
determining the initial velocity. The uncatalysed rates were deter-mined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were pre-pared in distilled deionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6 h at room temperature prior to assay in order to allow for the formation of the E I com-plex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng Prusoff equa-tion, as reported earlier21–23, and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier21,24.
3. Results and discussion
3.1. ChemistryStarting from the benzaldehyde derivative 1, the synthesis of the key intermediate 7 was reported earlier by our groups1. Briefly,
the synthesis of 7-amino-3H-1,2-benzoxathiepine 2,2-dioxide (7) was started with a Wittig reaction in which 5-nitro-salicylic alde-hyde 1 was converted to the corresponding mono-olefin 2 in 65% yield (Scheme 1). Treatment of compound 2 with allyl sulphonyl chloride (3) provided the bisolefin 4 in 65% yield. In the next step, the olefin metathesis reaction with Ru-catalyst 5 was employed, leading to the conversion of compound 4 to 7-nitro-3H-1,2-benzoxathiepine 2,2-dioxide 6 in 96% yield. The nitro derivative 6 was thereafter reduced with iron in acidic medium to
the corresponding amine 7 in nearly quantitative yield (98%). The key intermediate 7 was subsequently reacted with a series of acyl chlorides to afford the desired compounds 8–17 in good to Table 1. Inhibition data of human CA isoforms CA I, II, IX and XII with 3H-1,2-benzoxathiepines 2,2-dioxide 8–17 using acetazolamide (AAZ) as a standard drug. O S OO 8-17 H N R O Cmpd R KI(nM) a,b
hCA I hCA II hCA IX hCA XII 8 CH3 >100 mM >100 mM 61.8 162.5 9 C6H5 >100 mM >100 mM 208.6 370.1 10 4-CH3-C6H4 >100 mM >100 mM 83.0 309.3 11 4-Br-C6H4 >100 mM >100 mM 353.3 140.7 12 2-I-C6H4 >100 mM >100 mM 45.4 643.7 13 2-Br-C6H4 >100 mM >100 mM 66.8 96.2 14 2-F-C6H4 >100 mM >100 mM 74.6 40.3 15 2-CF3-C6H4 >100 mM >100 mM 19.7 8.7 16 thien-2-yl >100 mM >100 mM 177.5 73.2 17 furan-2-yl >100 mM >100 mM 210.1 134.4 AAZ – 250 12 25 5.7 a
Mean from three different assays, by a stopped flow technique (errors were in the range of ±5–10% of the reported values).
b Incubation time 6 h. O SO2 O2N O OH O2N OH O2N O O2N S O O O SO2 H2N 1 2 4 6 7 O SO2 H N O R 8-17 (i) SO2Cl 3 5 (iii) (iv) (ii) R= Me ; ; ; ; 8 (70%) 9 (72%) 10 (73%) 11 (59%) Me ; ; ; ; 12 (88%) 13 (82%) 14 (79%) 15 (87%) Br N N Ru Cl Cl PCy3 Ph 5 (v) Br I F F3C ; . 16 (81%) 17 (81%) S O
Scheme 1. Reagents and conditions: (i) MePPh3Br, tBuOK, THF, RT, 18 h, 65%; (ii) Net3, CH2Cl2, 0C to RT, 4 h, 57%; (iii)5, toluene, 70C, 4 h, 96%; (iv) Fe, AcOH,
EtOH, H2O, 75C, 1 h, 98%; (v) RCOCl, Net3, CH2Cl2, 0C to RT, 4 h.
excellent yields (see Experimental for details). The nature of moi-eties R was chosen in such a way to assure chemical diversity. Apart R¼ Me in compound 8, the remaining derivatives 9–17 incorporated aromatic or heterocyclic moieties, such as phenyl, 2-or 4-substituted phenyls, thienyl and furyl. We found out in previ-ous papers1–3 that aryl or hetaryl moieties on the sulfocoumarin, homosulfocoumarin or coumarin ring6 systems lead to com-pounds with an effective inhibition profile against CA isoforms of pharmacologic interest, such as the tumour-associated ones CA IX and XII.
3.2. Carbonic anhydrase inhibition
The obtained homosulfocoumarins 8–17 were investigated for their CA inhibitory properties by using a stopped-flow CO2 hydrase assay20and four human CA isoforms (hCA I, II, IX, and XII) known to be drug targets1(Table 1).
As seen from data ofTable 1, derivatives 8–17 did not signifi-cantly inhibit the cytosolic isoforms hCA I and II, similar to other homosulfocoumarins, sulfocouamrins or coumarins investigated earlier1–8. On the other hand, the transmembrane, tumour-associ-ated isoforms hCA IX and XII were inhibited by all these com-pounds in the nanomolar range. For hCA IX the KIs were in the range of 19.7–353.3 nM whereas for hCA XII in the range of 8.7–643.7 nM (Table 1). The nature of the R moiety on the carbox-amide functionality greatly influenced the inhibitory power. For hCA IX/XII the optimal substitution was that present in compound 15, 2-trifluoromethylphenyl, whereas the one leading to the least effective inhibitor was the one with 4-bromophenylcarboxamide moiety (compound 9) for hCA IX and 2-iodophenylcarboxamide (compound 12) for hCA XII. Overall, all these new homosulfocou-marins act as isoform IX/XII selective CAIs over hCA I and II, which is highly desirable for these new chemotypes with enzyme inhibi-tory properties.
4. Conclusions
A series of 3H-1,2-benzoxathiepine 2,2-dioxides incorporating 7-acylamino moieties were obtained by an original procedure start-ing from 5-nitrosalicylaldehyde which was treated with propenyl-sulfonyl chloride followed by cyclisation through a Wittig reaction of the bis-olefin intermediate. The new derivatives, belonging to the homosulfocoumarin chemotype, were assayed as inhibitors of the zinc metalloenzyme CA. Four pharmacologically relevant human (h) isoforms were investigated, the cytosolic hCA I and II, and the transmembrane, tumour-associated hCA IX and XII. No relevant inhibition of hCA I and II was observed, whereas some of the new derivatives were effective, low nanomolar hCA IX/XII inhibitors, making them of interest for investigations in situations in which the activity of these isoforms is overexpressed, such as hypoxic tumours, arthritis or cerebral ischaemia.
Disclosure statement
The author(s) do not declare any conflict of interest.
Funding
This work was partly supported by ERA.Net RUS plus joint pro-gramme project THIOREDIN (State Education Development Agency of Republic of Latvia) and the Russian Foundation for
Basic Research [project grant 18–515-76001] under ERA.Net RUS plus joint programme grant RUS_ST2017-309.
ORCID
Claudiu T. Supuran http://orcid.org/0000-0003-4262-0323
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